SMARTer Mouse TCR a/b Profiling Kit User Manual

SMARTer Mouse TCR a/b Profiling Kit User Manual Cat. Nos. 634402, 634403 & 634404 (102016) 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: tech@clontech.com United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.565.6999 Page 1 of 21

Table of Contents I. Introduction... 3 II. List of Components... 6 III. Additional Materials Required... 8 IV. General Considerations... 9 A. Requirements for Preventing Contamination... 9 B. General Requirements... 9 C. Sample Recommendations... 9 D. Sample Requirements... 10 V. Protocols... 10 A. Protocol: First-Strand cdna Synthesis... 10 B. Protocol: TCR a/b Amplification and Sequencing Library Generation by Semi-Nested PCR... 13 C. Protocol: Purification of Amplified Libraries Using Agencourt AMPure XP Beads... 16 D. Protocol: Library Validation... 18 VI. References... 19 Appendix A: Illumina HT Indexes... 19 Appendix B: Guidelines for Library Sequencing and Data Analysis... 21 Table of Figures Figure 1. Protocol overview and timeline.... 3 Figure 2. Schematic of technology and workflow for SMARTer Mouse TCR a/b Profiling Kit.... 5 Figure 3. Example electropherogram results for TCR-α and TCR-β libraries.... 19 Figure 4. Percentage of bases called per cycle from a typical MiSeq TCR-α and TCR-β profiling run... 21 Table of Tables Table 1. Sample Preparation Guidelines... 11 Table 2. Cycling Guidelines Based on Amount of Starting Material... 13 Table 3. TCR a/b Mouse Indexing Primer Set HT for Illumina Index Sequences... 20 Customer Service/Ordering tel: 800.662.2566 (toll-free) fax: 800.424.1350 (toll-free) web: http://www.takarabio.com e-mail: orders@clontech.com Contact Us Technical Support tel: 800.662.2566 (toll-free) fax: 800.424.1350 (toll-free) web: http://www.takarabio.com e-mail: tech@clontech.com Page 2 of 21

I. Introduction T-Cell Receptor Profiling Using SMART Technology The SMARTer Mouse TCR a/b Profiling Kit (Cat. Nos. 634402, 634403 & 634404) enables users to analyze T-cell receptor (TCR) diversity from total RNA samples and whole cells. This kit is designed to work with a range of RNA input amounts (depending on the sample type), and has been shown to generate high-quality libraries from as little as 10 ng to 500 ng of total RNA obtained from spleen, thymus, PBMCs, or from 1,000 to 10,000 purified T cells. As the name suggests, the kit can be used to generate data for both alpha- and beta-chain diversity, either in the same experiment or separately. Included in the kit are primers that incorporate Illumina specific adaptor sequences during cdna amplification. The protocol generates indexed libraries that are ready for sequencing on Illumina platforms. Starting Material: RNA or Purified T cells Begin with 10 ng 500 ng of RNA or 1,000 10,000 cells First-Strand cdna Synthesis (Section V.A) Hands-on time = 30 min, reaction time = 1 hr PCR 1 (Section V.B.1) Hands-on time = 10 min, reaction time = 30 min PCR 2 (Section V.B.2) Hands-on time = 15 min, reaction time = 30 min* Library Purification (Section V.C) Hands-on time = ~1 hr Figure 1. Protocol overview and timeline. * depends on number of cycles The SMARTer Mouse TCR a/b Profiling Kit leverages SMART technology (Switching Mechanism at 5 End of RNA Template) and employs a 5 RACE-like approach to capture complete V(D)J variable regions of TCR transcripts (Figure 2). First-strand cdna synthesis is dt-primed (TCR dt Primer) and performed by the MMLVderived SMARTScribe Reverse Transcriptase (RT), which adds non-templated nucleotides upon reaching the 5 end of each mrna template. The SMART-Seq v4 Oligonucleotide anneals to these non-templated nucleotides, and serves as a template for the incorporation of an additional sequence of nucleotides into the firststrand cdna by the RT. (This is the template-switching step.) This additional sequence referred to as the SMART sequence serves as a primer-annealing site for subsequent rounds of PCR, ensuring that only sequences from full-length cdnas undergo amplification. Page 3 of 21

Following reverse transcription, two rounds of PCR are performed in succession to amplify cdna sequences corresponding to variable regions of TCR-α and/or TCR-β transcripts: The first PCR uses the first-strand cdna as a template and includes a forward primer with complementarity to the SMART sequence (SMART Primer 1), and a reverse primer that is complementary to the constant (i.e., non-variable) region of TCR-α and/or TCR-β (TCRa and/or TCRb Mouse Primer 1); both reverse primers may be included in a single reaction if analysis of both TCR subunit chains is desired. By priming from the SMART sequence and the constant region, the first PCR specifically amplifies the entire variable region and a considerable portion of the constant region of TCR-α and/or TCR-β cdna. The second PCR takes the product from the first PCR as a template, and uses semi-nested primers (TCR Primer 2 and TCRa and/or TCRb Mouse Primer 2) to amplify the entire variable region and a portion of the constant region of TCR-α and/or TCR-β cdna. As in PCR 1, either or both TCR subunit chains may be amplified in a single reaction. The forward and reverse primers include adapter and index sequences that are compatible with the Illumina sequencing platform and allow for multiplexing of up to 96 samples in a single flow-cell lane. Following post-pcr purification, size selection, and quality analysis, the library is ready for sequencing. Page 4 of 21

Figure 2. Schematic of technology and workflow for SMARTer Mouse TCR a/b Profiling Kit. Page 5 of 21

II. List of Components SMARTer Mouse TCR a/b Profiling Kit User Manual The SMARTer Mouse TCR a/b Profiling Kit consists of the SMARTer Mouse TCR a/b Profiling Kit Components (not sold separately), the TCR a/b Mouse Indexing Primer Set HT for Illumina (not sold separately), and PrimeSTAR GXL SP DNA Polymerase. These components have been specifically designed to work together and are optimized for this particular protocol. Please do not make any substitutions. The substitution of reagents in the kit and/or a modification of the protocol may lead to unexpected results. The reaction number for each kit specifies the number of libraries (TCR-α alone, TCR-β alone, or TCR-α and TCR-β combined) that can be generated with the indexing primers supplied. SMARTer Mouse TCR a/b Profiling Kit Cap color 634402 (12 rxns) SMARTer Mouse TCR a/b Profiling Kit Components (Not sold separately. Storage conditions are listed below for Package 1 and Package 2.) Package 1 (Store at 70 C.) 634403 (48 rxns) 634404 (96 rxns) Control RNA (1 µg/µl) Clear 5 µl 5 µl 5 µl Package 2 (Store at 20 C. Once thawed, store 10X Lysis Buffer at 4 C and store Elution Buffer at room temperature. Continue to store all other reagents at 20 C.) SMART-Seq v4 Oligonucleotide (48 µm) Pink 12 µl 48 µl 96 µl TCR dt Primer (12 µm) Blue 24 µl 96 µl 192 µl 5X Ultra Low First-Strand Buffer Red 48 µl 192 µl 384 µl SMARTScribe Reverse Transcriptase (100 U/µl) Purple 24 µl 96 µl 192 µl Nuclease-Free Water Clear 1 ml 3 x 1 ml 2 x 4 ml RNase Inhibitor (40 U/µl) White 30 µl 120 µl 240 µl 10X Lysis Buffer Clear/ * 230 µl 920 µl 1.85 ml Elution Buffer (10 mm Tris-Cl, ph 8.5) Clear 1.7 ml 1.7 ml 2 x 1.7 ml TCRa Mouse Primer 1 (12 µm) Orange 12 µl 48 µl 96 µl TCRb Mouse Primer 1 (12 µm) Orange 12 µl 48 µl 96 µl SMART Primer 1 (12 µm) Green 6 µl 24 µl 48 µl PrimeSTAR GXL SP DNA Polymerase (1.25 U/µl) White 50 µl 100 µl 5X PrimeSTAR GXL SP Buffer White 0.5 ml 1 ml dntp Mixture (2.5 mm each) White 200 µl 400 µl PrimeSTAR GXL SP DNA Polymerase Package 3 (Store at 20 C.) RF220Q PrimeSTAR GXL SP DNA Polymerase (1.25 U/µl) White 200 µl 5X PrimeSTAR GXL SP Buffer Clear 2 x 1 ml dntp Mixture (2.5 mm each) White 800 µl * Depending on the product size, 10X Lysis Buffer is packaged in a 1.5 ml tube with a clear cap (Cat. Nos. 634402 and 634403) or in an 8-ml bottle (Cat. No. 634404). Page 6 of 21

SMARTer Mouse TCR a/b Profiling Kit, continued SMARTer Mouse TCR a/b Profiling Kit User Manual Cap color 634402 (12 rxns) 634403 (48 rxns) 634404 (96 rxns) TCR a/b Mouse Indexing Primer Set HT for Illumina (Not sold separately. Store at 20 C. For details, see Appendix A) TCRa Mouse Primer 2 Reverse HT Index 1 (ar1; 12.5 µm) Red 12 µl 12 µl 12 µl TCRa Mouse Primer 2 Reverse HT Index 2 (ar2; 12.5 µm) Red 12 µl 12 µl TCRa Mouse Primer 2 Reverse HT Index 3 (ar3; 12.5 µm) Red 12 µl 12 µl TCRa Mouse Primer 2 Reverse HT Index 4 (ar4; 12.5 µm) Red 12 µl 12 µl TCRa Mouse Primer 2 Reverse HT Index 5 (ar5; 12.5 µm) Red 12 µl TCRa Mouse Primer 2 Reverse HT Index 6 (ar6; 12.5 µm) Red 12 µl TCRa Mouse Primer 2 Reverse HT Index 7 (ar7; 12.5 µm) Red 12 µl TCRa Mouse Primer 2 Reverse HT Index 8 (ar8; 12.5 µm) Red 12 µl TCRb Mouse Primer 2 Reverse HT Index 1 (br1; 12.5 µm) Red 12 µl 12 µl 12 µl TCRb Mouse Primer 2 Reverse HT Index 2 (br2; 12.5 µm) Red 12 µl 12 µl TCRb Mouse Primer 2 Reverse HT Index 3 (br3; 12.5 µm) Red 12 µl 12 µl TCRb Mouse Primer 2 Reverse HT Index 4 (br4; 12.5 µm) Red 12 µl 12 µl TCRb Mouse Primer 2 Reverse HT Index 5 (br5; 12.5 µm) Red 12 µl TCRb Mouse Primer 2 Reverse HT Index 6 (br6; 12.5 µm) Red 12 µl TCRb Mouse Primer 2 Reverse HT Index 7 (br7; 12.5 µm) Red 12 µl TCRb Mouse Primer 2 Reverse HT Index 8 (br8; 12.5 µm) Red 12 µl TCR Primer 2 Forward HT Index 1 (F1; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 2 (F2; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 3 (F3; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 4 (F4; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 5 (F5; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 6 (F6; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 7 (F7; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 8 (F8; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 9 (F9; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 10 (F10; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 11 (F11; 12.5 µm) Blue 12 µl 12 µl 12 µl TCR Primer 2 Forward HT Index 12 (F12; 12.5 µm) Blue 12 µl 12 µl 12 µl Storage Conditions Store Control RNA at 70 C. Store 10X Lysis Buffer at 20 C. Once thawed, the buffer can be stored at 4 C. Store Elution Buffer at 20 C. Once thawed, the buffer can be stored at room temperature. Store all other reagents at 20 C. Page 7 of 21

III. SMARTer Mouse TCR a/b Profiling Kit User Manual Additional Materials Required The following reagents and materials are required but not supplied. They have been validated to work with this protocol. Please do not make any substitutions because you may not obtain the expected results: Single-channel pipettes: 10 µl, 20 µl, and 200 µl Eight-channel pipettes (recommended): 20 µl and 200 µl Filter pipette tips: 2 µl, 20 µl, and 200 µl Minicentrifuge for 1.5-ml tubes Minicentrifuge for 0.2-ml tubes or strips For PCR Amplification & Validation: One dedicated thermal cycler used only for first-strand cdna synthesis (Section V.A) One dedicated thermal cycler used only for library amplification by PCR (Section V.B) For validation using the Advanced Analytical Fragment Analyzer: High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, Cat. No. DNF-474; Section V.D) For validation using the Agilent 2100 Bioanalyzer: DNA 1000 Kit (Agilent, Cat. No. 5067-1504; Section V.D) Nuclease-free thin-wall PCR tubes or strips (0.2-ml PCR 8-tube strip; USA Scientific, Item No. 1402-4700) Nuclease-free low-adhesion 1.5-ml tubes (USA Scientific, Item No. 1415-2600) or LoBind tubes (Eppendorf, Cat. No. 022431021) For SPRI (Solid Phase Reversible Immobilization) Bead Purifications (Section V.C): Agencourt AMPure XP PCR purification kit used to purify amplified libraries (5-ml size: Beckman Coulter Item No. A63880; 60-ml size: Beckman Coulter Item No. A63881) NOTE: SPRI beads need to come to room temperature before the container is opened. Therefore, we recommend aliquoting the beads into 1.5-ml tubes upon receipt, and then refrigerating the aliquots. Individual tubes can be removed for each experiment, allowing them to come to room temperature more quickly (~30 min). This will also decrease the chances of bead contamination. Mix well to disperse the beads before adding them to your reactions. The beads are viscous, so pipette slowly. 100% ethanol (molecular biology grade) Magnetic separation device for small volumes used to purify amplified libraries For 8-tube strips*: Magnetic Separator - PCR Strip, Takara Bio, Cat. No. 635011 For 96-well plates: Magnetic Stand-96, Thermo Fisher, Cat. No. AM10027 Optional, depending on the choice of magnetic separation device (96-well format): - 96-well V-bottom Plate (500 µl; VWR, Cat. No. 47743-996) - MicroAmp Clean Adhesive Seal (Thermo Fisher, Cat. No. 4306311) - Low-speed benchtop centrifuge for a 96-well plate * Please visit www.clontech.com/rna-seq-tips for a video tutorial on how to construct your own efficient magnetic separation device from rare earth bar magnets and a tip rack. Page 8 of 21

IV. General Considerations SMARTer Mouse TCR a/b Profiling Kit User Manual A. Requirements for Preventing Contamination Before you set up the experiment, make sure you have two physically separated work stations: A PCR Clean Work Station for all pre-pcr experiments that require clean room conditions, e.g., first-strand cdna synthesis (Section V.A). NOTES: - The PCR Clean Work Station must be located in a clean room with positive air flow, as contamination may occur very easily. Once contamination occurs it can be difficult to remove. - Strictly obey clean room operation rules. A second work station located in the general laboratory where you will perform PCR (Section V.B) and measure library concentration (Section V.D). B. General Requirements The success of your experiment depends on the quality of your input RNA. Prior to cdna synthesis, please make sure that your RNA is intact and free of contaminants. Avoid using heparin for blood sample collection, as it can inhibit downstream enzymatic steps such as cdna synthesis and PCR. The assay is very sensitive to variations in pipette volume, etc. Please make sure all your pipettes are calibrated for reliable delivery, and make sure nothing is attached to the outside of the tips. All lab supplies related to cdna synthesis need to be stored in a DNA-free, closed cabinet. Reagents for cdna synthesis need to be stored in a freezer/refrigerator that has not previously been used to store PCR amplicons. Add enzymes to reaction mixtures last, and thoroughly incorporate them by gently pipetting the reaction mixture up and down. Do not increase (or decrease) the amount or concentration of any reaction component. The amounts and concentrations have been carefully optimized for the included reagents and protocol. If you are using this protocol for the first time, we strongly recommend that you perform negative and positive control reactions to verify that kit components are working properly. C. Sample Recommendations Total RNA Extraction The performance of the SMARTer Mouse TCR a/b Profiling Kit is dependent on the quality of the RNA starting material. IMPORTANT: Input RNA should be free from poly A+ carrier RNA that will interfere with oligo(dt)-primed cdna synthesis. When choosing a purification kit, ensure that it is appropriate for your sample amount. There are several commercially available kits designed to purify total RNA from extremely small samples (e.g., Takara Bio offers the NucleoSpin RNA XS kit for purification of RNA from 100 cells; Cat. No. 740902.10). Evaluation of RNA Quality After RNA extraction, if your sample size is not limiting, we recommend evaluating total RNA quality using the Agilent RNA 6000 Pico Kit (Cat. No. 5067-1513). Refer to the manufacturer s instructions about how to use the Agilent RNA 6000 Pico Kit. Page 9 of 21

D. Sample Requirements SMARTer Mouse TCR a/b Profiling Kit User Manual Total RNA This protocol has been optimized for cdna synthesis starting from 10 ng of total RNA. However, if your RNA sample is not limiting, we recommend that you start with more total RNA. (The protocol works well with inputs up to 500 ng; however, 10 ng 250 ng is the optimal range.) Purified total RNA should be in nuclease-free water. Cells This protocol has been used successfully to generate cdna starting from 1,000 10,000 intact, cultured cells. It cannot be used with cells that have undergone fixation. IMPORTANT: Cells should be washed and then resuspended in PBS that is free of Mg 2+ and Ca 2+ ions prior to lysis in order to remove the culture media. The presence of media can interfere with first-strand synthesis. V. Protocols NOTE: Please read the entire protocol before starting. This protocol has been optimized for generation of sequencing-ready libraries for TCR repertoire profiling from 10 ng of input RNA. Due to the sensitivity of the protocol, the input material (RNA or cells) needs to be collected and purified under clean-room conditions to avoid contamination. The whole process of cdna synthesis should be carried out in a PCR Clean Work Station under clean-room conditions. A. Protocol: First-Strand cdna Synthesis (Perform in PCR Clean Work Station) First-strand cdna synthesis (from RNA) is primed by the TCR dt Primer and uses the SMART-Seq v4 Oligonucleotide for template switching at the 5 end of the transcript. For this protocol, you will need the following components: 10X Lysis Buffer (tube with a clear cap or a bottle, depending on product size), RNase Inhibitor (white cap), Nuclease-Free Water (clear cap), Control RNA (clear cap), TCR dt Primer (blue cap), 5X Ultra Low First-Strand Buffer (red cap), SMART-Seq v4 Oligonucleotide (pink cap), SMARTScribe Reverse Transcriptase (purple cap). 1. Thaw the 5X Ultra Low First-Strand Buffer at room temperature. Thaw all the remaining reagents needed for first-strand cdna synthesis (except the enzyme) on ice. Gently vortex each reagent to mix and spin down briefly. Store all but the 5X Ultra Low First-Strand Buffer on ice. NOTE: The 5X Ultra Low First-Strand Buffer may form precipitates. Thaw this buffer at room temperature and vortex before using to ensure all components are completely in solution. 2. Prepare a stock solution of 10X Reaction Buffer by mixing the 10X Lysis Buffer with the RNase Inhibitor as indicated below (scale up as needed): 19 μl 10X Lysis Buffer (tube with a clear cap or a bottle, depending on product size) 1 μl RNase Inhibitor (white cap) 20 μl Total volume Mix briefly, then spin down. NOTE: Lysis Buffer contains a detergent. It is critical to avoid bubbles when mixing. Page 10 of 21

3. See Table 1 for guidelines on setting up reactions for your control and test samples. Prepare each sample (10.5 µl total volume) in individual 0.2-ml RNase-free PCR tubes or in an 8-well strip: Purified Total RNA: If you are working with purified total RNA, transfer 1 9.5 μl to a 0.2-ml RNase-free PCR tube. Bring the volume to 9.5 μl with nuclease-free water. Add 1 μl of 10X Reaction Buffer. Cells: If you are working with cells, isolate cells in validated media, wash and resuspend in Mg 2+ /Ca 2+ -free PBS, and transfer to a 0.2-ml RNase-free PCR tube. Bring the volume to 9.5 μl with nuclease-free water. Add 1 μl of 10X Reaction Buffer. Gently vortex or pipette to mix the sample. Incubate at room temperature for 5 min. See Section IV.D for more information on working with cells. Table 1. Sample Preparation Guidelines Components Negative Control Positive Control Test Sample 10X Reaction Buffer (from Step 2) 1 µl 1 µl 1 µl Nuclease-Free Water 9.5 µl Up to 8.5 µl Up to 8.5 µl Diluted Control RNA* 1 9.5 µl Sample 1 9.5 µl Total Volume 10.5 µl 10.5 µl 10.5 µl *The Control RNA is supplied at a concentration of 1 μg/μl. It should be diluted in Nuclease-Free Water with RNase Inhibitor (1 μl RNase Inhibitor in a final volume of 50 μl of water) to match the concentration of your test sample. Perform serial dilutions on the Control RNA to obtain the appropriate concentration. We have tested this protocol extensively using the PCR cycling conditions below with 10 ng of input Control RNA. 4. Place the samples on ice, and add 2 μl of the TCR dt Primer (blue cap) to each sample. Mix well by gently vortexing and then spin the tube(s) briefly to collect the contents at the bottom of the tube. 5. Preheat the thermal cycler to 72 C. 6. Incubate the tubes at 72 C in the preheated, hot-lid thermal cycler for 3 min. NOTE: Prepare your Master Mix (Step 7) while your tubes are incubating. The enzyme will be added just before use (Step 9). Steps 10 11 below are critical for first-strand cdna synthesis and should not be delayed after completing Step 7. 7. Prepare enough Master Mix for all the reactions, plus 10% of the total reaction mix volume, by combining the following reagents in the order shown at room temperature. Per reaction: 4 μl 5X Ultra Low First-Strand Buffer (red cap)* 1 μl SMART-Seq v4 Oligonucleotide (pink cap) 0.5 μl RNase Inhibitor (white cap) 2 μl SMARTScribe Reverse Transcriptase (purple cap)** 7.5 μl Total volume added per reaction * The 5X Ultra Low First-Strand Buffer should be thawed at room temperature and vortexed gently to remove any cloudiness in the buffer before using. ** Add the reverse transcriptase to the Master Mix just prior to use, making sure to gently mix the reverse transcriptase tube without vortexing before adding it. 8. Mix the Master Mix well by gently pipetting up and down and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube. 9. Immediately after the 3 min incubation at 72 C (Step 6), place the samples on ice for 2 min. Page 11 of 21

10. Reduce the temperature of the thermal cycler to 42 C. SMARTer Mouse TCR a/b Profiling Kit User Manual 11. Add 7.5 μl of the Master Mix to each reaction tube. Mix the contents of each tube by pipetting gently, and spin briefly to collect the contents at the bottom of each tube. 12. Place the tubes in a thermal cycler with a heated lid, preheated to 42 C. Run the following program: 42 C 45 min 70 C 10 min 4 C forever STOPPING POINT: The tubes can be stored at 4 C overnight. Page 12 of 21

B. Protocol: TCR a/b Amplification and Sequencing Library Generation by Semi-Nested PCR (Perform Steps 1 3 in a PCR Clean Work Station) IMPORTANT: This PCR protocol has been optimized based on an initial input of 10 ng of total RNA (isolated from mouse spleen) into the RT reaction described in the previous protocol. Optimal parameters may vary for different templates, cell types, thermal cyclers, and sample amounts. We strongly recommend trying a range of cycle numbers to determine the minimum number of cycles necessary to obtain the desired yield for your sample and conditions. Table 2 provides guidelines for optimization, depending on the amount of total RNA or the number of cells used for first-strand cdna synthesis. Table 2. Cycling Guidelines Based on Amount of Starting Material Analysis of TCR-α and TCR-β sequences using both primer sets: Input Type Input Amount Number of PCR 1 Cycles Number of PCR 2 Cycles Control RNA 10 100 ng 21 18 20 Control RNA 100 250 ng 21 16 18 Control RNA 250 500 ng 21 16 Purified T cells 1,000 10,000 cells 21 20 Analysis of TCR-α or TCR-β using the respective primer set: Input Type Input Amount Number of PCR 1 Cycles Number of PCR 2 Cycles Control RNA 10 100 ng 21 16-18 Control RNA 100 250 ng 21 16 Control RNA 250 500 ng 21 16 Purified T cells 1,000 10,000 cells 21 20 1. PCR 1 This PCR selectively amplifies TCR sequences from the first-strand cdna generated with the previous protocol. SMART Primer 1 anneals to the SMART sequence (incorporated during firststrand cdna synthesis) and adds the Illumina Read 2 sequence. TCRa Mouse Primer 1 and/or TCRb Mouse Primer 1 anneal to sequences in the constant regions of TCRa and TCRb cdna, respectively. For this protocol, you will need the following components: 5X PrimeSTAR GXL SP Buffer (white [634402 & 634403] or clear [634404] cap), dntp Mixture (white cap), SMART Primer 1 (green cap), TCRa Mouse Primer 1 and/or TCRb Mouse Primer 1 (orange cap), PrimeSTAR GXL SP DNA Polymerase (white cap), and Nuclease-Free Water (clear cap). 1. Thaw all reagents needed for PCR (except the enzyme) on ice. Gently vortex each reagent tube to mix and spin down briefly. Store on ice. 2. Prepare enough PCR Master Mix for all the reactions, plus 10% of the total reaction mix volume. Combine the following reagents (amounts are for 1 reaction) in the order shown: Page 13 of 21

10 μl 5X PrimeSTAR GXL SP Buffer (white [634402 & 634403] or clear [634404] cap) 4 μl dntp Mixture (white cap) 0.5 μl SMART Primer 1 (green cap) 0.5 μl TCRa Mouse Primer 1 and/or TCRb Mouse Primer 1 (orange cap)* 1 μl PrimeSTAR GXL SP DNA Polymerase (white cap) 14 μl Nuclease-Free Water (clear cap) 30 μl Total volume added per reaction * If using TCRa Mouse Primer 1 in combination with TCRb Mouse Primer 1, mix 1 μl of TCRa Mouse Primer 1 with 1 μl of TCRb Mouse Primer 2 and use 0.5 μl of the mixture (scale up as required). NOTE: Remove the PrimeSTAR GXL SP DNA Polymerase from the freezer, gently mix the tube without vortexing, and add to the Master Mix just before use. Mix the Master Mix well by vortexing gently and spin the tube briefly to collect the contents at the bottom of the tube. 3. Add 30 μl of PCR Master Mix to each tube containing 20 µl of the first-strand cdna product from Section V.A. Mix well and briefly spin to collect the contents at the bottom of the tube(s). IMPORTANT: Transfer the samples from the PCR Clean Work Station to the general lab. All downstream processes should be performed in the general lab. 4. Place the tube(s) in a preheated thermal cycler with a heated lid and run the following program: PCR 1 95 C 1 min 21 cycles: 98 C 10 secs 60 C 15 secs 68 C 45 secs 4 C forever STOPPING POINT: The tubes may be stored at 4 C overnight. 2. PCR 2 This PCR further amplifies sequences corresponding to full-length TCR variable regions and adds Illumina HT sequencing adapters using a semi-nested approach. The TCR Primer 2 Forward HT Index primers anneal to a sequence added by SMART Primer 1 and add Illumina P7-i7 index sequences. The nested TCRa and/or TCRb Mouse Primer 2 Reverse HT Index primers anneal to sequences in TCR constant regions that are internal to the sequences bound by TCRa Mouse Primer 1 and TCRb Mouse Primer 1, and add both the Illumina Read 1 sequence and P5-i5 index sequences. IMPORTANT: Different combinations of Forward HT indexes (F1 F12) and Reverse HT indexes (R1 R8) must be used for each sample if samples are to be multiplexed on a single flow cell. See Appendix A for further details. For this protocol, you will need the following components: 5X PrimeSTAR GXL SP Buffer (white [634402 & 634403] or clear [634404] cap), dntp Mixture (white cap), TCR a/b Mouse Indexing Primer Set HT for Illumina (blue and red caps), PrimeSTAR GXL SP DNA Polymerase (white cap), and Nuclease-Free Water (clear cap). Page 14 of 21

1. Thaw all reagents needed for PCR (except the enzyme) on ice. Gently vortex each reagent tube to mix and spin down briefly. Store on ice. 2. Prepare enough PCR Master Mix for all the reactions, plus 10% of the total reaction mix volume. Combine the following reagents (amounts are for 1 reaction) in the order shown: 10 μl 5X PrimeSTAR GXL SP Buffer (white [634402 & 634403] or clear [634404] cap) 4 μl dntp Mixture (white cap) 0.5 μl TCRa and/or TCRb Mouse Primer 2 Reverse HT Index (red cap)* 1 μl PrimeSTAR GXL SP DNA Polymerase (white cap) 33 μl Nuclease-Free Water (clear cap) 48.5 μl Total volume added per reaction * If using both TCRa and TCRb Mouse Primer 2 Reverse HT Index primers in combination, mix 1 μl of TCRa Mouse Primer 2 Reverse HT Index with 1 μl of TCRb Mouse Primer 2 Reverse HT Index and use 0.5 μl of the mixture (scale up as required). NOTE: Remove the PrimeSTAR GXL SP DNA Polymerase from the freezer, gently mix the tube without vortexing, and add to the Master Mix just before use. Mix the Master Mix well by vortexing gently and spin the tube briefly to collect the contents at the bottom of the tube. 3. For each reaction, add 48.5 μl of PCR Master Mix to a clean 0.2 ml tube. 4. Add 1 μl of PCR product from PCR 1 to each tube. 5. Add 0.5 μl of the appropriate TCR Primer 2 Forward HT Index primer (blue cap) to each sample. Mix well and briefly spin to collect the contents at the bottom of the tube(s). 6. Place the tube(s) in a preheated thermal cycler with a heated lid and run the following program: PCR 2 (similar to PCR 1, but with reduced cycle numbers) 95 C 1 min 16 20 cycles*: 98 C 10 secs 60 C 15 secs 68 C 45 secs 4 C forever * Consult Table 2 (above) for PCR cycle number guidelines. STOPPING POINT: The tubes may be stored at 4 C overnight. Page 15 of 21

C. Protocol: Purification of Amplified Libraries Using Agencourt AMPure XP Beads The TCR sequencing library is size-selected and purified using AMPure XP beads. This approach involves two rounds of size selection, which together remove primers, primer dimers, and PCR products containing undersized or oversized inserts. In the first round, fragments larger than ~900 bp are immobilized on beads and removed from the supernatant. In the second round, the supernatant is added to fresh beads, which immobilize fragments within the desired size range of ~400 900 bp. The beads are then washed with 80% ethanol and fragments are eluted with Elution Buffer. This approach preserves library yield and complexity while maximizing the relevance and consistency of downstream sequencing results. NOTES: - Aliquot AMPure XP beads into 1.5-ml tubes upon receipt in the laboratory. - Before each use, bring bead aliquots to room temperature for at least 30 min and mix well to disperse. - Prepare fresh 80% ethanol for each experiment. You will need 400 µl per sample. - You will need a magnetic separation device for 0.2-ml tubes, strip tubes, or a 96-well plate. For this protocol, you will need the following components: Agencourt AMPure XP PCR Purification beads, 80% ethanol (made fresh), a magnetic separation device, and Elution Buffer (clear cap). 1. Vortex AMPure XP beads until evenly mixed, then add 22.5 μl of beads to each sample. 2. Mix thoroughly by pipetting the entire volume up and down at least 10 times. NOTE: The beads are viscous; pipette the entire volume, and push it out slowly. Do not vortex. Vortexing will generate bubbles, making subsequent handling of the beads difficult. 3. Incubate at room temperature for 8 min to let the DNA bind to the beads. 4. Briefly spin the samples to collect the liquid from the side of the tube or sample well. Place the samples on the magnetic separation device for ~5 min or longer, until the liquid appears completely clear and there are no beads left in the supernatant. The time required for the solution to clear will depend on the strength of the magnet. NOTE: Ensure that the solution is completely clear, as any bead carryover will decrease the efficiency of size selection. There is no disadvantage in separating the samples for >5 min. 5. While the reaction tubes are sitting on the magnetic separation device, use a pipette to transfer the supernatant (which contains your library) to clean PCR tubes. 6. Remove the tubes containing the beads from the magnetic separation device and discard them. Add 10 μl of AMPure XP beads to each tube containing supernatant. NOTE: Ensure that the beads are fully resuspended before use. If the beads appear to have settled at the bottom of the tube, gently vortex to ensure that they are completely mixed. 7. Mix thoroughly by pipetting the entire volume up and down at least 10 times. NOTE: The beads are viscous; pipette the entire volume, and push it out slowly. Do not vortex. Vortexing will generate bubbles, making subsequent handling of the beads difficult. 8. Incubate at room temperature for 8 min to let the DNA bind to the beads. Page 16 of 21

9. Place the tubes on the magnetic separation device for ~10 min or until the solution is completely clear. 10. While the tubes are sitting on the magnetic separation device, remove the supernatant with a pipette and discard it. (The library is now bound to the beads.) 11. Keep the tubes on the magnetic separation device. Add 200 μl of freshly made 80% ethanol to each sample without disturbing the beads, to wash away contaminants. Wait for 30 seconds and use a pipette to carefully remove the supernatant containing contaminants. The library will remain bound to the beads during the washing process. 12. Repeat the ethanol wash (Step 11) once. OPTIONAL: Briefly spin the tubes (~2,000g) to collect the remaining liquid at the bottom of each tube. Place the tubes on the magnetic separation device for 30 seconds, then remove all remaining liquid with a pipette. 13. Let the sample tubes rest open on the magnetic separation device at room temperature for ~2 2.5 min until the pellet appears dry and is no longer shiny. You may see a tiny crack in the pellet. NOTE: Be sure to dry the pellet only until it is just dry. The pellet will look matte with no shine. If you under-dry the pellet, ethanol will remain in the sample wells. The ethanol will reduce your library recovery rate and ultimately your yield. Allow the plate to sit at room temperature until the pellet is dry. If you over-dry the pellet, there will be cracks in the pellet. It will take longer than 2 min to rehydrate (Step 15) and may reduce library recovery and yield. Visit www.clontech.com/rna-seq-tips to view examples of moist, dry, and overly dry pellets. 14. Once the bead pellet has dried, remove the tubes from the magnetic separation device and add 17 μl of Elution Buffer to cover the pellet. Remove the samples from the magnetic separation device and mix thoroughly by pipetting up and down to ensure complete bead dispersion. NOTE: Be sure that the beads are completely resuspended. The beads can sometimes stick to the sides of the tube. 15. Incubate at room temperature for at least 5 min to rehydrate. 16. Briefly spin the samples to collect the liquid from the side of the tube or sample well. Place the samples back on the magnetic separation device for 2 min or longer, until the solution is completely clear. NOTE: There may be a small population of beads that do not pellet against the magnet during incubation. Pipet these unpelleted beads up and down to resuspend them with the supernatant, and then pipet them towards the magnet where the rest of the beads have already pelleted (without disrupting the existing pellet). Continue the incubation until there are no beads left in the supernatant. 17. Transfer clear supernatant containing the purified TCR library from each tube to a nuclease-free, low-adhesion tube. Label each tube with sample information and store at 20 C. STOPPING POINT: The samples may be stored at 20 C indefinitely. Page 17 of 21

D. Protocol: Library Validation To determine whether library production, purification, and size selection were successful, we recommend analyzing and validating final libraries using an Advanced Analytical Fragment Analyzer and the High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, Cat. No. DNF-474) or the Agilent 2100 Bioanalyzer and the DNA 1000 Kit (Agilent, Cat. No. 5067-1504). Please refer to the corresponding user manuals for detailed instructions. 1. Aliquot 1 μl of diluted library for validation using the Advanced Analytical Fragment Analyzer. (Libraries were diluted 1:5 for the examples shown on the next page.) Alternatively, aliquot 1 μl of undiluted library for validation using the Agilent 2100 Bioanalyzer. 2. Compare the results for your samples with Figure 3 to verify whether each sample is suitable for further processing. Successful library production and purification should yield no product for negative control reactions, and a broad peak spanning 650 bp to 1,150 bp, with a maximum between ~700 bp and ~800 bp for positive controls and samples containing TCR RNA. The position and shape of electropherogram peaks will vary depending on whether TCR-α and/or TCR-β sequence fragments are included in the library, the nature of the RNA sample, and the analysis method. Libraries consisting of either TCR-α or TCR-β sequence fragments yield electropherogram maxima that tend toward the upper and lower ends of the ~700 800 bp size spectrum, respectively. For some sample types (including RNA obtained from a single T-cell clonotype), analysis of libraries containing both TCR-α and TCR-β sequence fragments may yield two discernable peaks in the electropherogram results. In general, electropherogram peaks obtained with the Fragment Analyzer tend to be sharper than those obtained with the Bioanalyzer. 3. Following validation, libraries are ready for sequencing on Illumina platforms. See Appendix B for sequencing guidelines. Page 18 of 21

Figure 3. Example electropherogram results for TCR-α and TCR-β libraries. Libraries containing TCR-α and TCR-β sequences were generated using the SMARTer Mouse TCR a/b Profiling Kit and 10 ng of RNA obtained from either a heterogeneous sample of spleen RNA or a monoclonal population of EL4 cells, respectively. Both libraries were produced using 21 amplification cycles for PCR 1 and 20 amplification cycles for PCR 2. Following purification and size selection, both libraries were analyzed on an Advanced Analytical Fragment Analyzer (Panels A and C) and an Agilent 2100 Bioanalyzer (Panels B and D). Peaks labeled LM and UM correspond to DNA reference markers included in each analysis. Panels A and B show broad peaks between ~650 1,150 bp and maximal peaks in the range of ~700 800 bp (typical results for a library generated from spleen RNA), analyzed on the Fragment Analyzer or Bioanalyzer, respectively. More distinct peaks representing TCR-α and TCR-β sequence fragments can be discerned when a library generated from EL4 RNA is analyzed on the Fragment Analyzer (Panel C), but this is not as evident when the same library is analyzed on the Bioanalyzer (Panel D). VI. References Bolotin, D. A. et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat. Methods 12, 380 381 (2015). Page 19 of 21

Appendix A: Illumina HT Indexes SMARTer Mouse TCR a/b Profiling Kit User Manual Unique combinations of Illumina indexes are required in order to discriminate between samples when sequencing a pool of two or more libraries on a single flow cell. Consult the Illumina literature (such as the TruSeq DNA Sample Preparation Guide) for appropriate pooling guidelines. The TCR Primer 2 Forward HT Index primers contain the Read 2 sequence and i7 indexes. These primers are labeled sequentially ( F1 F12 ), in correspondence with Illumina indexes D701 D712. In addition to gene-specific sequences derived from the constant regions of Mouse TCR-α and TCR-β, the TCRa and TCRb Mouse Primer 2 Reverse HT Index primers contain the Read 1 sequence and i5 indexes. Depending on the kit size, 1 8 different TCRa and TCRb Mouse Primer 2 Reverse HT Index primers containing unique i5 indexes are included for amplification of TCRa and TCRb, respectively. These primers are labeled sequentially ( Mouse ar1 Mouse ar8 or Mouse br1 Mouse br8 ), in correspondence with Illumina indexes D501 D508. The pairwise combination of 12 unique i7 indexes with 8 unique i5 indexes allows for multiplexing of up to 96 samples in a single flow cell lane. Table 3. TCR a/b Mouse Indexing Primer Set HT for Illumina Index Sequences. TCR Primer 2 Forward HT Index TCR a/b Mouse Primer 2 Reverse HT Index Primer ID (cap label) Illumina ID Index sequence Primer ID (cap label) Illumina ID Index sequence F1 D701 ATTACTCG Mouse ar1 or Mouse br1 D501 TATAGCCT F2 D702 TCCGGAGA Mouse ar2 or Mouse br2 D502 ATAGAGGC F3 D703 CGCTCATT Mouse ar3 or Mouse br3 D503 CCTATCCT F4 D704 GAGATTCC Mouse ar4 or Mouse br4 D504 GGCTCTGA F5 D705 ATTCAGAA Mouse ar5 or Mouse br5 D505 AGGCGAAG F6 D706 GAATTCGT Mouse ar6 or Mouse br6 D506 TAATCTTA F7 D707 CTGAAGCT Mouse ar7 or Mouse br7 D507 CAGGACGT F8 D708 TAATGCGC Mouse ar8 or Mouse br8 D508 GTACTGAC F9 D709 CGGCTATG F10 D710 TCCGCGAA F11 D711 TCTCGCGC F12 D712 AGCGATAG Page 20 of 21

Appendix B: Guidelines for Library Sequencing and Data Analysis Samples should be pooled to a final pool concentration of 4 nm. We recommend diluting the pooled libraries to a final concentration of 13.5 pm, including a 5 10% PhiX Control v3 (Illumina, Cat. No. FC-110-3001) spike-in. While not essential, the addition of the PhiX control increases the nucleotide diversity and thus aids in high-quality data generation. Sequencing should be performed on an Illumina MiSeq sequencer using the 600-cycle MiSeq Reagent Kit v3 (Illumina, Cat. No. MS-102-3003) with paired-end, 2 x 300 base pair reads. Upon completion of a sequencing run, data can be analyzed on a variety of platforms, including MiXCR (Bolotin et al. 2015) freely available software hosted on BaseSpace, Illumina's cloud computing environment for next-generation sequencing. MiXCR was specifically designed to process sequencing data obtained for T- and B-cell receptor repertoires, and can calculate clonotype frequencies from raw sequence inputs consisting of paired- or single-end reads. MiXCR also accounts for sequence quality, corrects PCR errors, and identifies germline hypermutations. While not required for analysis with MiXCR and many other publicly available software packages, the initial 35 bases of Read 2 (which includes the SMART primer sequence) may need to be trimmed from raw sequence reads prior to downstream analyses (see Figure 4). Figure 4. Percentage of bases called per cycle from a typical MiSeq TCR-α and TCR-β profiling run. Note the SMART primer sequence at the beginning of Read 2, which may need to be trimmed prior to data analysis. Cycles 301 315 are the index reads. Notice to Purchaser Our products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without prior written approval of Your use of this product is also subject to compliance with any applicable licensing requirements described on the product s web page at http://www.takarabio.com. It is your responsibility to review, understand and adhere to any restrictions imposed by such statements. 2016 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at http://www.takarabio.com. This document has been reviewed and approved by the Quality Department. Page 21 of 21